Chromic picolinate treatment

ABSTRACT

The administration of chromic picolinate, preferably in the form of chromic tripicolinate, as a prophylactic or therapeutic agent for controlling various blood serum parameters. In particular, the administration is for controlling blood serum lipid levels, including the lowering of undesirably high blood serum LDL-cholesterol levels and the raising of blood serum HDL-cholesterol levels. Additionally, the administration is for controlling undesirable high blood serum glucose levels, this being particularly applicable for the treatment of maturity-onset diabetes. Additionally, chromic picolinate, preferably in the form of chromic tripicolinate, is administered to facilitate uptake of amino acids by skeletal muscle. Thus chromic picolinate can act as an effective anabolic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.07/326,590, filed Mar. 31, 1989, now U.S. Pat. No. 5,087,623, which is acontinuation-in-part of U.S patent application Ser. No. 07/200,390entitled "Chromic Picolinate Treatment," filed May 31, 1988, nowabandoned which is a continuation-in-part of U.S. patent applicationSer. No. 07/126,588, entitled "Chromic Picolinate Treatment," filed Nov.30, 1987 now abandoned.

FIELD OF THE INVENTION

The field of the invention is nutritional trace elements and, morespecifically, chromium compounds.

DESCRIPTION OF RELATED ART AND INTRODUCTION TO THE INVENTION

Heart disease, more specifically, atherosclerotic heart disease, is aleading cause of death in the United States. High serum cholesterol hasbeen unequivocally established as a major risk factor for heart disease.Notwithstanding this knowledge, millions of Americans and others aroundthe world have cholesterol levels in the high risk range. Often thesecholesterol levels are resistant to conventional dietary modification,i.e., a reduction in dietary saturated fat and cholesterol.

Diabetes is known to afflict at least 10 million Americans, and millionsmore may have the disease without knowing it. In the form of thisdisease known as maturity-onset diabetes (as opposed to juvenilediabetes), the pancreas often continues to secrete normal amounts ofinsulin, but this insulin is ineffective in preventing the symptoms ofdiabetes which include hyperglycemia, impaired carbohydrate metabolism,glycosuria, and decreased insulin sensitivity.

Chromium is a nutritionally essential trace element, as are iron,iodine, copper and zinc, among others. The essentiality of chromium inthe diet was established in 1959 by Schwarz, as cited in PresentKnowledge in Nutrition, page 571, Fifth Edition (1984, The NutritionFoundation, Washington, D.C., U.S.A.). This reference, and all othersreferred to herein, are hereby incorporated by reference. Schwarz placedmice on a highly purified diet that excluded chromium. The mice became"glucose intolerant" and, thus, were no longer able to metabolizeglucose (blood sugar) normally. The mice developed symptoms identical tonon-insulin dependent diabetes. When chromium was restored to the dietvia brewer's yeast, normal glucose metabolism was restored. Schwarznamed the unknown chromium-dependent molecule responsible for therestoration of normal glucose metabolism the "Glucose Tolerance Factor"("GTF").

Chromium depletion is characterized in animals by disturbance ofglucose, lipid, and protein metabolism and by a shortened life span.Chromium is essential for optimal insulin activity in all knowninsulin-dependent systems. Boyle et al., Southern Med. J. 70: p.1449-1453 (1977) at 1450. Insufficient dietary chromium has been linkedboth to maturity-onset diabetes and to cardiovascular diseases.

The principle energy sources for the body are glucose and fatty acids.Chromium depletion results in biologically ineffective insulin and acompromised glucose metabolism, and the body must then rely primarily onlipid metabolism to meet energy requirements. Such individuals produceexcessive amounts of acetyl-CoA and ketone bodies. Part of theaccumulated acetyl-CoA is diverted to increased cholesterolbiosynthesis, thus resulting in hypercholesterolemia. Diabetes mellitusis manifested in large measure by glycosuria, hypercholesterolemia, andoften, ketoacidosis. The accelerated atherosclerotic process seen indiabetics, it has been noted, is associated with hypercholesterolemia.Boyle et al., supra.

Studies conducted by the U.S. Department of Agriculture show that thedietary chromium intake of most individuals, though, is considerablyless than the suggested safe and adequate intake. It has also beenconcluded that the suboptimal dietary intake of chromium is exacerbatedby increased chromium losses due to stress and, furthermore, thatcertain refined foods which include simple sugars enhance chromiumlosses. Supplementation of chromium to normal individuals has beenreported to lead to improvements in glucose tolerance, serum lipidconcentrations, including high-density lipoprotein cholesterol, insulinand insulin binding. Anderson, Clin. Physiol. Biochem. 4:31-41 (1986).Supplemental chromium in the trivalent form, e.g., chromic chloride, isassociated with improvements of risk factors associated withmaturity-onset diabetes and cardiovascular diseases.

Chromium, in the form of GTF, is known to function as a co-factor forthe hormone insulin. It binds to insulin and potentiates many, andperhaps all, of the functions of insulin. Boyle et al, supra. Thesefunctions include, but are not limited to, the regulation ofcarbohydrate and lipid (fat) metabolism. Present Knowledge In Nutrition,supra, at p. 573-77.

Introducing inorganic chromium compounds per se into individuals is,however, not particularly beneficial. Chromium must be convertedendogenously into an organic complex or it must be consumed as abiologically active molecule. Only about 0.5% of ingested inorganicchromium is assimilated in the body. "Recommended Dietary Allowances,"page 160, 9th Revised Edition (1980, The National Academy of Sciences).Only 1-2% of most chromium is assimilated while 10-25% of chromium inbrewer's yeast is assimilated. Present Knowledge In Nutrition, supra atp. 582. This is probably due to the fact that, as we have discovered,much of the chromium in brewer's yeast is in the form of GTF.

The results of a study have been published describing an investigationinto the effects of brewer's yeast on lipoprotein cholesterolconcentrations in a group of male volunteers. The study was initiatedbased upon reports that chromium, or brewer's yeast (which is rich inthe chromium-containing compound GTF), could lower serum totalcholesterol. Riales, "Chromium in Nutrition and Metabolism" p. 199(Shapcott & Hubert, Eds.) (1979). It was concluded that there was anincrease in HDL-cholesterol concentration, the beneficial form ofcholesterol. It has also been reported that groups of the populationwith coronary artery disease have significantly lower serum chromiumconcentrations than groups of the population having normallyunobstructed arteries. Newman et al., Clin. Chem.. 24:541 (1978).

In U.S. Pat. No. 4,315,927, issued on Feb. 16, 1982, to Gary W. Evansfor "Dietary Supplementation With Essential Metal Picolinates," Evansdescribes the discovery that when selected essential metals areadministered to mammals as exogenously synthesized coordinationcomplexes of picolinic acid, they are directly available for absorptioninto the system without competition from other metals. That patentdescribes one object of the invention to be the ability to provide acomposition and method for selectively supplementing the essentialmetals in the diets of humans and mammalian animals and for facilitatingabsorption of these metals by the intestinal cells. Other describedobjects of the invention include the correction of predetermined metaldeficiencies in mammals, the elimination of symptoms of thosedeficiencies without concurrently reducing the assimilated levels ofother essential metals, and the administration of trace metals in asafe, physiological form which is simple to produce and economicallyfeasible to distribute on a commercial basis. The exogenouslysynthesized essential metal coordination complexes of picolinic acid(pyridine-2-carboxylic acid) for use in the invention of U.S. Pat. No.4,315,927 are characterized by the following structural formula:##STR1## Wherein M represents the metallic cation and n is equal to thecation's valence. When M^(n) ("chromic" as used in this application) isC⁺³ and n=3 then the compound is chromic tripicolinate. Other chromiumpicolinates could include M=Cr⁺³ and n=2 ("chromic dipicolinate") or n=1("chromic monopicolinate") , each with additional amions present. Thepatent states that the particular trace elements of interest are zinc,iron, and chromium, although the patent also mentions copper, cobalt,and manganese. The patent further describes methods for preparation ofmetal picolinates, including zinc, copper, iron and chromium.

Plasma cholesterol and triglycerides are transported in lipoproteins.Hyperlipoproteinemias are conditions in which the concentrations ofthese cholesterol- or triglyceride-carrying lipoproteins exceed thenormal limit. Clinical concern arises because an elevated concentrationof lipoproteins can accelerate the development of atherosclerosis, withthe secondary possibilities of thrombosis and infarction. About half ofthe deaths in the United States are a result of such events Recent worksuggests that reduction of lipoprotein concentrations in plasma candiminish the increased risk of atherosclerosis that accompanieshyperlipoproteinemia.

The hyperlipoproteinemias have been designated as either primary orsecondary. Secondary hyperlipoproteinemias are complications of a moregeneralized metabolic disturbance, such as diabetes mellitus orexcessive intake of ethanol. The primary hyperlipoproteinemias aretypically caused either by an inherited single-gene defect (monogenichyperlipoproteinemias) or a combination of multiple subtle geneticfactors that act together with environmental ones (multifactorial orpolygenic hyperlipoproteinemias).

Much circumstantial evidence exists that treatment ofhyperlipoproteinemia will diminish or prevent atheroscleroticcomplications. For example, numerous populations studies have shown thatan elevated concentration of total cholesterol or LDL- cholesterol inplasma constitutes a major risk factor for the occurrence ofatherosclerotic events. In the case of monogenic disorders, familystudies have documented a markedly increased risk of vascular diseaseamong affected members. Until recently, however, treatment ofhyperlipoproteinemia was a controversial issue, primarily because thelowering of plasma lipids had not been shown to prolong life or diminishthe clinical complications of atherosclerosis.

In 1984, results of the Lipid Research Clinic's Coronary PrimaryPrevention trial, a multi-center, randomized, double-blind study,provided strong evidence that a reduction in plasma concentrations ofLDL-cholesterol can reduce the risk of coronary heart disease. LipidResearch Clinic's Program, "The lipid research clinic's coronary primaryprevention trial results," J.A.M.A. 251:351-374 (1984). Analysis of therelationship between reduction of cholesterol and coronary heart diseasesuggests that the incidence of coronary heart disease in patients withhyperlipoproteinemia would be reduced by nearly 50% for individuals whoachieved a 25% reduction in plasma total cholesterol or a 35% fall inplasma LDL-cholesterol.

In view of the incidence of heart disease in the United States andaround the world, and the high incidence of undesirable bloodcholesterol and triglyoeride levels which can lead to heart disease andother disorders, it would be desirable to have a simple way of loweringundesirable blood lipid levels. Described herein is the use of chromicpicolinate compounds, preferably chromic tripicolinate, to prevent andtreat undesirable levels of blood cholesterol, as well as to controlsymptoms associated with maturity-onset diabetes.

It is also known that anabolic agents, such as some steroids, can beused by individuals to promote an increase in lean body mass, probablyby decreasing serum amino acid levels. Anabolic steroids, though, areknown to have serious side effects when taken over prolonged periods. Itwould therefore also be desirable to have an alternative to agents suchas anabolic steroids which can promote an increase in lean body mass.

SUMMARY OF THE INVENTION

Ingested chromium can combine with picolinic acid, which is made in thebody, to form chromic picolinate. As noted, however, chromium is noteasily metabolized. We have discovered that chromic tripicolinate occursin brewer's yeast and has Glucose Tolerance Factor activity. We havealso discovered that chromic picolinate, preferably chromictripicolinate, can be used to prevent and relieve undesirably highlevels of lipids, including LDL-cholesterol, glucose, and productsformed as a result of high levels of glucose, such as glycosylatedhemoglobin in the blood. Furthermore, we have also discovered thatchromic picolinate, preferably chromic tripicolinate, can be used as atherapeutic agent to relieve some of the symptoms of maturity-onsetdiabetes and reduce the need for exogenous insulin and/or otherhypoglycemic agents, such as the sulfonylureas. Additionally it has beendiscovered that chromic picolinate, preferably chromic tripicolinate,can be used as an anabolic agent to increase lean body mass. It isexpected that chromic picolinate, and in particular chromictripicolinate, can be used in the dosage range corresponding to about 10to about 500 micrograms of chromium per day for the above indications.

DRAWINGS

Embodiments of the invention are described with reference to thedrawings in which:

FIG. 1 illustrates the design of the human study of EXAMPLE 3 (describedbelow).

FIGS. 2 through 7, illustrate the values of various parameters measuredduring the study of EXAMPLE 3, and show the beneficial effect of chromictripicolinate on serum lipids and glucose.

FIG. 8 and 9 illustrate the values of various parameters measured duringanother study on human subjects (EXAMPLE 4 below) which also show thebeneficial effects of chromic tripicolinate

FIGS. 10-13 illustrate further test results from an additional humanstudy (EXAMPLE 5 below) which show the anabolic effect of chromictripicolinate.

DETAILED DESCRIPTION OF THE INVENTION

We have identified picolinic acid as a natural component of brewer'syeast and have discovered that chromic tripicolinate is a highlyassimilable, biologically-active form of chromium that binds to insulinand insulin receptors. We have further discovered that, in fact, chromictripicolinate is a biologically-active form of chromium or, statedanother way, it has Glucose Tolerance Factor activity.

Chromic tripicolinate, because of its assimilability and because it willbind to insulin and insulin receptors, is capable of enhancing thenormal functions of insulin, which include:

a. reduction of elevated serum cholesterol;

b. improvement of glucose tolerance; and

c. reduction in fasting hyperglycemia.

The "recommended" or "safe and adequate" intake of chromium has beenestablished by the National Academy of Sciences as 50-200 microgramsdaily. Recommended Dietary Allowances, pp. 159-161, Ninth Edition (1980,National Academy of Sciences). Because chromic tripicolinate isassimilated more readily, as we have shown below, and will be moreeffective for the uses disclosed herein than other compounds ofchromium, a lesser amount of chromic tripicolinate may be required forsuch uses i.e., the prophylactic function of preventing or reducingserum lipids, total serum cholesterol and LDL-cholesterol, and thetherapeutic function of alleviating the symptoms of maturity-onsetdiabetes.

EXAMPLE 1

This Example illustrates the far greater increased rate of absorbtion ofchromic tripicolinate over an inorganic chromium salt (chromicchloride), in a mammal.

Protocol

In this example, chromic tripicolinate was first synthesized bycombining 3.66 grams of picolinic acid (Sigma Chemical, St. Louis) and2.66 grams of chromic chloride with 20 ml. of deionized water. Thesolution was heated with stirring for 15 minutes after which thecontents were placed in a refrigerator overnight to obtain maximumcrystalization. The supernatant was removed by vacuum filtrationfollowed by recrystalization from hot deionized water. To synthesizechromic (chromium-51) tripicolinate, 0.12 micromoles recrystalizedpicolinic acid was added to a solution containing 0.03 micromoles ofchromic (Cr⁵¹) chloride in 5.0 ml. acetate buffer, pH 5.0. The solutionwas stirred for 30 minutes at 90° C., cooled and subsequently reheatedfor 30 minutes at 90° C. The solution was cooled to room temperaturebefore use.

Each of four male Sprague-Dawley rats weighing 125-150g was given anoral dose of 150 ng. chromic (Cr-51) picolinate in 0.51 ml. by gavagetube. An identical quantity of Cr-5I as chloride in acetate buffer wasalso administered to each of four rats. The rats had been fed standardlab chow prior to an 18 hr. fast and were given free access to food andwater after administration of the isotopes. One hundred twenty hoursafter oral administration of the two forms of labeled chromium, each ofthe rats was killed by exsanguination after which the liver and kidneyswere removed and cut into 1.0 g portions for assay of radioactivitycontent.

Results

The results obtained are listed in the following Table 1.

It will be seen from Table 1 that chromic tripicolinate is absorbed at arate about 4 times greater than that of chromic chloride.

                  TABLE 1                                                         ______________________________________                                                           Total radioactivity                                                           (CPM) in tissue                                            Form of isotape      Liver   Kidney                                           ______________________________________                                        Chromic (Cr-51) Chloride                                                                            30050   39030                                           Chromic (Cr-51) Picolinate                                                                         115075  157525                                           ______________________________________                                    

EXAMPLE 2 Protocol

In a toxicity experiment, rats weighing a mean of 223.4 g were given anoral suspension of 100 mg, 300 mg and 500 mg chromic tripicolinate inwater. No deaths occurred and no visible effects were seen with anythese oral doses. The tests were stopped at 500 mg because it wasimpossible to obtain a workable suspension beyond this level.

Results

These results indicate that the oral lethal dose of chromictripicolinate is in excess of 2.2 g/Kg. Stated in human terms, a 70 Kg154 pound) human would have to ingest a dose greater than 156 grams ofchromic tripicolinate before any effects would be observed.

Three studies on human subjects were performed, as described in Examples3 to 5 below, which illustrate the operation of the present invention.

EXAMPLE 3 Protocol

Subjects for the study were obtained after screening a group ofvolunteers. Thirty-two volunteers age 25-80 with total cholesterollevels of 5.7-8.3 mmoles/l (220-320 mg/dl) were initially chosen. Nosubject had a history of hypothyroid disease, renal failure, liverdisease, diabetes mellitus, known familial lipid disorder, alcohol ordrug abuse, bleeding disorders, multiple allergies or any other seriousmedical illness and none were pregnant at the outset. None of thesubjects was using beta blockers, thiazide diuretics, steroids, chromiumsupplements or any investigational drugs. The volunteers were requestednot to alter dietary or exercise habits during the course of the study.Informed consent was obtained and the subjects were paid a stipend fortheir participation.

Chromium tripicolinate was synthesized by first dissolving 2.66 gCrCl₃.6 H₂ O in 25 ml warm (60° C.), deionized water. Thereafter, 3.69 gpicolinic acid (Sigma Chemical, St. Louis, Mo., U.S.A.) was added andthe contents were stirred with gentle heating until the solution turnedpurple and crystals began to form. The solution was then left standingat 4° C. overnight. The supernatant was then discarded and the crystalswere airdried at room temperature. Analysis of the crystals indicatedthat the complex consisted of 3 moles picolinate/1 mole Cr⁺³, MW=418.

For the double-blind, cross-over study, subjects were divided randomlyinto two treatment groups by a non-participant. During each half of thestudy participants ingested gelatin capsules that contained eitherchromic tripicolinate or a placebo. The chromic tripicolinate supplementcontained 1.6 mg chromium tripicolinate (200 micrograms Cr⁺³) mixed with5 mg calcium phosphate. The placebo contained only 5 mg calciumphosphate. The study consisted of two 42-day periods with a 14-dayperiod off capsules between treatments. Compliance by subjects wasmonitored by capsule count. The foregoing study protocol is illustratedin FIG. 1.

At the beginning, after three weeks and at the end of each period, heartrate, blood pressure, weight and temperature were measured and afasting-blood sample was drawn. Serum parameters measured included totalcholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides,apolipoprotein A₁ and apolipoprotein B. Serum concentrations ofcholesterol and triglycerides were determined with an Olympus 500Automated Colormetric System (Olympus Instrument Co., Los Angeles,Calif., U.S.A.). HDL-cholesterol was assayed with an enzymeheparin-manganese chloride precipitation method (Albers et al., ClinChem., 1978; 24:853-856). LDL-cholesterol was estimated by the method ofFriedwald (Friedwald, Clin Chem., 1972; 18:499-502). Concentrations ofapolipoprotein A₁ and apolipoprotein B in serum were assayed with anephelometric immunoassay (Naito, J. Clin. Immunol.. 1986; 9:110-120)using a Boehringer laser kit.

Results

For statistical analysis of the measurements made in this study, thevalues obtained at the beginning and at the end of each 42-day periodwere used. Because each subject served as its own control, paired ttests were used to identify statistically significant differencesbetween mean values of changes and differences in changes. Student's ttest and p values were determiend with the program WORMSTAT™ (availablefrom Small Business Computers, New England, Conn., U.S.A.). The sametype of statistical analysis was performed on the results of EXAMPLE 5below. In all three studies of EXAMPLES 3, 4, and 5, p values less than0.05 were considered statistically significant.

The mean age of the women in the study was 5 years greater than the malepopulation (FIG. 2). Weight is reasonably correlated with height for thesubjects in the study, as seen in FIG. 3. The inference is that thesubjects who were studied appear to be representative of normal healthypopulation. PG,18

Of the original 32 participants in the study of EXAMPLE 3, 28 subjectscompleted the study. Two participants moved away, one became pregnant,and another (a 51 year old female) developed "hot flashes," elevatedcholesterol levels and started menopause. Two subjects of EXAMPLE 3,developed mouth ulcers, one patient was on placebo and the other onchromium supplement. Three subjects developed a fine rash about theirforearms; two subjects on placebo, one subject on chromiumsupplementation. No other abnormalities were noted.

The results obtained after analysis of the serum cholesterol and serumapolipoprotein fractions are illustrated in FIGS. 4-7. As shown in FIGS.4-6, total cholesterol, LDL-cholesterol and apolipoprotein B were allsignificantly decreased (p=0.007, p=0.015, and p=0.003, respectively)during the first 6-week period in subjects who began the study with thechromic tripicolinate supplement. No significant alterations (p>0.05)were observed in any of these serum parameters during the last 6-weekperiod when the subjects were ingesting the placebo.

No significant change (p>0.05) was observed in either serum totalcholesterol or LDL-cholesterol during the first 6-week period among thesubjects who began the study with the placebo supplement. However, bothtotal cholesterol and LDL-cholesterol decreased significantly (p=0.009and p=0.003, respectively) during the final 42 days while these subjectswere ingesting the chromic tripicolinate supplement (FIG. 4 and FIG. 5).In the group that began the study with the placebo, apolipoprotein Bincreased slightly, but not significantly (p=0.058) during the first 8weeks and then decreased, but not significantly (p=0.072), aftersupplementation with chromic tripicolinate (FIG. 6). Apolipoprotein A₁was significantly elevated (p=0.047) during the first 6-week period inthe subjects that started the study with the chromic tripicolinatesupplement. No significant change (p<0.05) was observed during the final42 days. Apolipoprotein A₁ in the placebo-first group increased, but notsignificantly (p=0.165), during the first six weeks, and then decreasedduring the off-capsule period. During the chromic tripicolinatesupplementation period, apolipoprotein A₁ was increased significantly(p=0.007) in these subjects (FIG. 7).

The consolidated results of the study are listed in Table 2 below. Whenthe results from the 28 subjects were combined, significant (p<0.001)reductions occurred with both total cholesterol and LDL-cholesterolconcentrations during the 42-day period that chromic tripicolinate wasingested. Total cholesterol concentration decreased 7% from 7.1 mmoles/lto 6.6 mmoles/l (276 mg/dl to 256 mg/dl) while LDL-cholesterol decreased10.5% from 5.7 mmoles/l to 5.1 mmoles/l (200 mg/dl to 178 mg/dl) duringtreatment with chromic tripicolinate. Of the final 28 subjects, 22 hadlower total cholesterol, 2 were unchanged and the remaining 4 hadcholesterol concentrations elevated less than 0.1 mmole/l (5 mg/dl).With LDL-cholesterol, 20 subjects had decreased concentrations, 3 wereunchanged and the others had slightly elevated concentrations ofLDL-cholesterol. The concentration of serum HDL-cholesterol increasedslightly but not significantly (p>0.05) during the period the subjectswere ingesting chromic tripicolinate.

While subjects were ingesting the placebo, slight but statisticallyinsignificant (p>0.05) elevations occurred in both serum totalcholesterol and LDL-cholesterol.

When the volunteers were treated with chromic tripicolinate, theconcentration of serum apolipoprotein B, the principal protein of theLDL-cholesterol fraction, was decreased significantly (p=0.001).Analysis of the values obtained from all 28 subjects showed a reductionof 16% from 155 mg/dl to 130 mg/dl. The principal protein of theHDL-cholesterol fraction, apolipoprotein A:, was significantly elevated(p=0.003) in the serum of subjects ingesting chromic tripicolinate.

During the period the subjects ingested the placebo, apolipoprotein Bwas elevated significantly (p>0.05). A slight, but not significant(p>0.05), elevation in apolipoprotein A₁ was also observed while thesubjects were supplemented with the placebo. No significant changes wereobserved in serum triglycerides, weight, blood pressure, temperature orheart rate with either chromic tripicolinate or placebo.

Despite the fact that the supplements were distributed randomly, theconcentration of each of the lipid parameters was consistently lower inthe subjects at the start of the placebo supplementation period. Bycoincidence, the 14 subjects that started the trial with the placebo hadserum lipids considerably lower than the 14 who started with chromictripicolinate. Thus, because the mean levels of 14 subjects wereinitially lower coupled with the fact that the other 14 started withlower lipid levels resulting from successful treatment with chromictripicolinate, the numerical means listed in Table 2 are less at thestart of treatment with the placebo. As shown in FIGS. 4-7, regardlessof the starting level, treatment with chromic tripicolinate producedsignificant changes.

                                      TABLE 2                                     __________________________________________________________________________    Serum lipid parameters after supplementation                                  with chromic tripicolinate and placebo                                                                   Treatment                                                   Chromic Tripicolinate                                                                           Placebo                                                                 Chng.             Chng.                                  Serum Fraction                                                                         Init  Final (P)   Init. Final (p)                                    __________________________________________________________________________    Cholesterol Total                                                             (mmole/l)                                                                              7.1 ± 1.0                                                                        6.6 ± 1.0                                                                        -0.5  6.8 ± 0.9                                                                        6.8 ± 1.0                                                                        +0.1                                   (mg/dl)  276 ± 40                                                                         256 ± 41                                                                         -19   262 ± 34                                                                         265 ± 38                                                                         +3                                                          p = .0003         p = .282                               LDL                                                                           (mmole/l)                                                                              5.7 ± 1.1                                                                        5.0 ± 1.0                                                                        -0.6  5.2 ± 0.9                                                                        5.3 ± 0.8                                                                        +0.1                                   (mg/cl)  200 ± 39                                                                         178 ± 35                                                                         -21   184 ± 31                                                                         188 ± 30                                                                         +4                                                          p = .0003         p = .106                               HDL                                                                           (mmol/l) 1.3 ± 0.4                                                                        1.4 ± 0.4                                                                        +0.1  1.4 ± 0.3                                                                        1.4 ±  0.4                                                                       +0                                     (mg/dl)  52 ± 16                                                                          55 ± 17                                                                          +3    53 ± 13                                                                          53 ± 17                                                                          +.7                                                         p = .118          p = .369                               Apolipoproteins                                                               Apo A (mg/dl)                                                                          147 ± 28                                                                         163 ± 31                                                                         +16   153 ± 26                                                                         164 ± 40                                                                         +11                                                         p = .003          p = 0.77                               Apo B (mg/dl)                                                                          155 ± 50                                                                         130 ± 45                                                                         -25   115 ± 32                                                                         129 ± 36                                                                         +14                                                         p = .001          p = .023                               Triglycerides                                                                 (mmol/l) 1.9 ± 0.7                                                                        1.9 ± 0.9                                                                        -0    1.9 ± 0.7                                                                        2.0 ± 1.0                                                                        +0.1                                   (mg/dl)  168 ± 65                                                                         167 ± 76                                                                         -1    165 ± 65                                                                         175 ± 88                                                                         +10                                                         p = .246          p = .151                               __________________________________________________________________________     All values are mean ± SD of 28 subjects                               

EXAMPLE 4 Protocol

Eleven subjects with adult onset diabetes mellitus (AODM), participatedin this study. These subjects had hemoglobin A₁ C concentrations greaterthan 9.0 and were on stable doses of oral hypoglycemic agents for fourmonths. The participants received 200 micrograms chromium in the form of1.6 mg chromium tripicolinate admixed with 5 mg of calcium phosphate,using the same protocol as in EXAMPLE 3 (FIG. 1). The dose of oral agentremained constant during the study.

The following parameters: Cholesterol, HDL, LDL, and hemoglobin A₁ Cwere tested initially, and at the end of each half of the study. Fastingblood sugars, blood pressure, weight and temperature were testedinitially; at three weeks; and at the end of the study.

Glycosylated hemoglobin was measured by a modification of the method ofClegg and Schroeder (Clegg, et al. J. Lab Clin. Med., 102:577-589,(1983)), using a Fast Hemoglobin Minicolumn, available from Isolab;Akron, Ohio, U.S.A.). Normal values are 5.5 to 7.5%. Fasting glucoseswere measured by finger stick glucometer readings (using an Accucheck IIfrom Boehringer Mannheim, Indianapolis, Ind., U.S.A.).

Split samples of cholesterol were sent to laboratories for analysis.Split samples of apolipoproteins were analyzed on a Beckman Arrayprotein analyzer that was standardized by the Center for DiseaseControl, Atlanta, Georgia, U.S.A., using Lipid Research ClinicGuidelines (see J. A. M. A. 251: 351, (1984) and J. A. M. A. 251: 365,(1984)).

Results

For EXAMPLE 4, summary statistics are expressed as the mean ± standarderror of the mean (SEM). Split-plot analyses of variance were used toassess treatment effects on the various parameters in the cross-overstudies of variance (see Hills, et al., Br. J. Clin. Pharmacol. 8:7-20,(1979)). Correlation analysis was performed with a linear regressioncurve-fitting program.

Analyses were performed on the following parameters for EXAMPLE 4:fasting blood sugars, hemoglobin A₁ C, cholesterol, LDL, HDL, weight,blood pressure, heart rate and temperature. Results are summarized inTable 3. There were no significant changes in weight, blood pressure,heart rate, and temperature. HDL and cholesterol trends were favorable,but with the small sample studies P was not <0.05. Hemoglobin A₁ Csignificantly decreased 12% in the treatment versus placebo groups (FIG.8). LDL significantly decreased by 8% (FIG. 9). All significant P valuesare <0.5.

                  TABLE 3                                                         ______________________________________                                        EXAMPLE 4: Effect of Supplemental Chromium                                    Tripicolinate on 11 Subjects With Adult Onset                                 Diabetes Mellitus Patients for the Parameters                                 Listed.                                                                                                 Cr Supplementation                                  Parameter  Placebo        6 Weeks                                             ______________________________________                                        Cholesterol                                                                              214.3 ± 12.2    211.4 ±                                                                             13.4                                   LDL        141.3 ± 9.4     139.5 ±                                                                             9.9                                    HDL        51.9 ±  6.0     52.3 ±                                                                              6.4                                    Weight     179.8 ± 9.4     179.6 ±                                                                             9.6                                    Blood Pressure                                                                           130.4/72.6 ±                                                                          5.4/3.4 132.9/72.4 ±                                                                        6.4/3.6                                Temperature                                                                              97.5 ±  .22     97.8 ±                                                                              .11                                    Heart Rate 70.2 ±  2.2     70.1 ±                                                                              2.1                                    Hemoglobin A.sub.1 C*                                                                    10.8 ±  0.1     10.4 ±                                                                              0.1                                    Fasting Bld                                                                              172.5 ± 12.9    162.2 ±                                                                             11.4                                   Sugar*                                                                        ______________________________________                                         Values are given as mean ± standard error of the mean (SEM)                Level of significance was p <0.05.                                            *Significant difference between placebo and chromium supplementation.    

EXAMPLE 5

This study was performed to evaluate the anabolic effect of chromictripicolinate in male subjects.

Protocol

At the start of the study, 10 male university students enrolled inweight training classes, were given, at random, a 40-day supply ofcapsules that contained either placebo (5 mg of calcium phosphate) orchromic tripicolinate (1.6 mg chromic tripicolinate admixed with 5 mg ofcalcium phosphate, which provided 200 ug of chromium). The identity ofthe capsules was known only to an individual who was not in any wayinvolved in the study. During the 40-day period, each of the volunteersfollowed a prescribed weight lifting protocol for a total of 3 hours perweek. Measurements taken at the beginning and again at the end of thestudy included various skin-fold thicknesses, biceps and calfcircumferences, and body weight. Percent body fat was derived from thesum of the triceps, subscapular and chest skinfolds. Lean body mass wasderived from anthropometrically-determined percent body fat:weight-(weight×percent body fat).

Results

Nine volunteers completed the study. Five of these were in the groupgiven the placebo.

In the group consuming the placebo, bicep circumference increasedsignificantly (P=0.017) 1.2±0.75 cm, while in the group given thechromic tripicolinate, the biceps circumference increased significantly(P=0.0056) 1,4±0.45 cm (see FIG. 10). In the placebo group, the calfcircumference increased 0.82±0.88 cm which was not significant(P=0.067), while in the chromic tripicolinate group calf circumferenceincreased significantly (P=0.029) 1.15±0.67 cm (see FIG. 11). In theplacebo group, body weight increased significantly (P=0.0465) 1.25±1.14kg, while the body weight of the group given chromic tripicolinateincreased significantly (P=0.0325) 2.18±1.34 kg (see FIG. 12). In theplacebo group, the calculated lean body mass increased only 0.04 kgwhich was not significant (P=0.464), while in marked contrast, thecalculated lean body mass of the group given chromic tripicolinateincreased significantly (P=0.017) 1.6 kg (see FIG. 13). Moreover, whenthe increase in lean body mass of the two groups was comparedstatistically by use of the independent variable Student's t test, theincrease seen in the group given chromic tripicolinate was significantlygreater (P=0.0198) than that of the placebo group.

The results obtained in EXAMPLE 3 demonstrate that chromic tripicolinateis an effective agent for the treatment of serum lipid disorders. Theresults of EXAMPLE 4 additionally demonstrate that chromic tripicolinateis effective in producing significant decreases in the levels of bothfasting glucose and glycosylated hemoglobin in diabetic subjects. Theresults of EXAMPLE 5 demonstrate that chromic tripicolinate can act asan anabolic agent, thus implying that it can facilitate the entry ofamino acids into skeletal muscles. Thus, chromic tripicolinate is abiologically active form of chromium. In addition, in the studies ofExamples 3 and 4, four of the six serum lipid parameters measured werealtered during the period the subjects ingested chromium. Totalcholesterol, LDL-cholesterol and the related transport proteinapolipoprotein B were decreased while apolipoprotein A₁, theHDL-cholesterol related protein, was elevated when the supplementcontained chromic tripicolinate. Because each of these parameters isassociated either directly or inversely to the onset of coronary arterydisease, chromic tripicolinate is an excellent agent for considerationin the treatment and prevention of hyperlipidemia. It will beappreciated that compositions could be used in place of chromictripicolinate which carry chromium ion and picolinate, provided thatfollowing administration to the individual, the chromium and picolinatecan combine to form chromic tripicolinate.

Total cholesterol is a treatable risk factor for coronary heart disease(Lipid Research Clinics Program, JAMA. 1984; 251:351. Lipid ResearchClinic's Program, JAMA, 1984; 251:365. Lipid Research Clinic's ProgramEpidemiology Committee Circulation, 1979; 60:127). During the 7.4 yearLipid Research Clinic's Coronary Primary Prevention Trial, an overallreduction of cholesterol of 8.5 percent was associated with a 19 percentdecrease in coronary artery heart disease. Furthermore, the initialstages of atherosclerosis are directly related to the level ofLDL-cholesterol (Newman et al., N. Engl J. Med., 1986; 314: 138-144).Both of these lipid fractions were decreased significantly after only 42days of supplementation with chromic tripicolinate. Total cholesterolwas reduced 7 percent while LDL-cholesterol was decreased 10 percentwith this treatment.

Several recent studies additionally demonstrate that specificlipoproteins are associated with heart disease (Avogaro, et al., Artery.1978; 4:385-394. Sniderman, et al., Proc. Natl. Acad Sci. USA, 1980;77:604-608. Riesen, et al., Atherosclerosis, 1980; 37:157-162. DeBacker,et al., Atherosclerosis, 1982; 42:197-203. Maciejko, et al., N. Engl. J.Med., 1983; 309:385-389. Schmidt, et al., Am. J. Cardiol., 1985;55:1459-1462. Kwiterovich, et al., Prev. Med., 1983; 12:815-834.Brunzell, et al., Arteriosclerosis, 1984; 4:79-83). In patients withdocumented coronary artery disease, elevated levels of apolipoprotein Bcoupled with reduced levels of apolipoprotein A₁ were observed. Duringour study, supplementation with chromic tripicolinate resulted in asignificant elevation of apolipoprotein A₁ and a significant reductionin apolipoprotein B. Further, some investigators have suggested thatlevels of apolipoprotein A₁ and apolipoprotein B are a better predictorof coronary artery heart disease than levels of total, LDL, or HDL,cholesterol. (Kwiterovich, et al., suora.; and Brunzell, et al.,suora.).

It is possible that higher doses of chromic tripicolinate than thoseused in EXAMPLE 3 above, may yield a greater reduction in totalcholesterol. Additionally, there would be the possible potentiatingeffect of combining chromium with diet or different lipid-loweringagents. Further, use of higher doses of chromic tripicolinate than thoseused in Examples 3 and 4 may also enhance the beneficial effects foundin those studies.

In order to reduce total serum cholesterol and LDL-cholesterol while atthe same time increasing high density lipoprotein (HDL) cholesterol, itis anticipated that the dosage of chromic tripicolinate administered toa patient will contain between about 10 and about 200 micrograms ofchromium, or more, with a dose of at least about 10 micrograms ofchromium being administered. When chromic tripicolinate is administeredas an anabolic agent, it is anticipated that the dosage will beapproximately within the same range. The chromic tripicolinate may beorally ingested by way of a pharmaceutically acceptable carrier in, forexample, tablet or capsule form. The chromic tripicolinate may typicallybe administered on a daily basis.

In order to reduce the requirement for insulin and/or diabetic drugs andreduce several important risk factors associated with maturity-onsetdiabetes, it is anticipated that the dosage range of chromiumadministered to a patient in the form of chromic tripicolinate will bebetween about 50 and about 500 micrograms, with a dose amount of atleast about 50 micrograms of chromium being administered.

For the purposes of this invention, the compounds of the invention maybe administered orally, parenterally, by inhalation spray, or bypercutaneous diffusion in formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous intravenous,intramuscular, and intraarterial injection and infusion techniques.Intraarterial and intravenous injection as used herein includesadministration through catheters. It is also anticipated that thesecompounds can be provided in the form of food additives.

Pharmaceutical compositions containing the active ingredient may be inany form suitable for the intended method of administration. When usedfor oral use, for example, tablets, lozenges, aqueous or oilsuspensions, dispersible powders or granules, emulsions, hard or softcapsules, syrups or elixirs may be prepared. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group including sweeteningagents, flavoring agents, coloring agents and preserving agents in orderto provide a palatable preparation. Tablets containing the chromictripicolinate in admixture with non-toxic pharmaceutically acceptableexcipients which are suitable for manufacture of tablets are acceptable.These excipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, such as maize starch,or alginic acid; binding agents such as starch, gelatin or acacia; andlubricating agents, such as magnesium stearate, stearic acid or talc.Tablets may be uncoated or may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearatealone or with a wax may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the chromic tripicolinatein admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include suspending agents, dispersing orwetting agents, one or more preservatives, one or more coloring agents,one or more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspension maycontain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by an added antioxidant such as ascorbicacid. Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Additional excipients,for example sweetening, flavoring and coloring agents, may also bepresent.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according toknown art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, such as a solution in 1,3-butanediol. Among theacceptable diluents and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile fixed oils may be employed conventionally as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid may likewise be used in the preparation of injectablepreparations.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The above description of the invention is set forth solely to assist inunderstanding the invention. It is to be understood that variations ofthe invention, including all equivalents now known or later developed,are to be considered as falling within the scope of the invention, whichis limited only by the hereafter appended claims.

We claim:
 1. A method for treating undesirable high levels of bloodserum lipids in an individual in need of such treatment, comprising theadministration of an effective blood serum lipid-reducing amount of acomposition consisting essentially of chromic tripicolinate.
 2. A methodfor treating undesirable high levels of blood serum lipids in anindividual in need of such treatment, comprising the administration ofan effective, blood serum lipid reducing amount of chromictripicolinate.
 3. A method for treating undesirable high levels of bloodserum lipids in an individual, comprising:a) evaluating the individualfor undesirable high levels of blood serum lipids; and b) then, when theevaluation indicates undesirable high levels of blood serum lipids,administering an effective, blood serum lipid reducing dose of chromictripicolinate.
 4. The method of claim 2 wherein said chromictripicolinate is in a pharmaceutically acceptable carrier and is orallyingested.
 5. The method of claim 2 wherein said chromic tripicolinate ispresent in a pharmaceutically acceptable carrier in a dose having atleast about 10 micrograms of chromium.
 6. The method of claim 2 whereinsaid chromic tripicolinate is present in a pharmaceutically acceptablecarrier in a dose having from about 10 to 200 micrograms of chromium. 7.The method of claim 3 wherein said chromic tripicolinate issubstantially pure.
 8. The method of claim 3 wherein said chromictripicolinate is substantially free of impurities from brewer's yeast.9. The method of claim 3 wherein said chromic picolinate is syntheticchromic tripicolinate.
 10. The method of claim 3 wherein said bloodserum lipids are serum triglycerides.
 11. The method of claim 3 whereinsaid blood serum lipids are total serum cholesterol.
 12. The method ofclaim 3 wherein said blood serum lipids are LDL-cholesterol.
 13. Themethod of claim 3 wherein a known dose of chromic tripicolinate isadministered on each of a plurality of days, the method additionallycomprising reevaluating the level of blood serum lipids.
 14. The methodof claim 13 wherein the administration of chromic tripicolinate iscontinued until the re-evaluation indicates a decrease in blood serumlipids.
 15. A method for treating undesirable high levels of blood serumlipids in an individual, comprising:a) evaluating the individual forundesirable high levels of blood serum lipids; and b) then, when theevaluation indicates undesirable high levels of blood serum lipids,administering an effective blood serum reducing amount of chromium ionand picolinate ion such the two can form chromic tripicolinate in theindividual.
 16. The method of claim 15 additionally comprisingre-evaluating the level of blood serum lipids, wherein chromium ion andpicolinate are administered on each of a plurality of days prior to there-evaluation until the re-evaluation indicates a decrease in bloodserum lipids.
 17. A method for increasing blood serum levels ofHDL-cholesterol in an individual in need thereof, comprising theadministration of an effective amount of chromic tripicolinate.
 18. Amethod for increasing an undesirable low level of blood serumHDL-cholesterol in an individual, comprising:a) evaluating theindividual for the undesirable low level of blood serum HDL-cholesterol;and b) then, when the evaluation indicates an undesirable low level ofblood serum HDL-cholesterol, administering an effective HDL-cholesterolincreasing dose of chromic tripicolinate.
 19. The method of claim 18wherein said dose of chromic tripicolinate is administered on each of aplurality of days, the method additionally comprising re-evaluating theblood serum HDL-cholesterol level, the administration of chromictripicolinate being continued until the re-evaluation indicates anincrease in blood serum HDL-cholesterol.
 20. A method for lowering bloodserum LDL-cholesterol in an individual in need of such reduction,comprising the administration, over a plurality of days, of chromictripicolinate in a dose having at least about 10 micrograms of chromiumper day.
 21. A method for lowering blood serum LDL-cholesterol in anindividual in need of such reduction, comprising:a) evaluating theindividual's blood serum LDL-cholesterol level; and b) thenadministering, over a plurality of days, a composition consistingessentially of chromic tripicolinate in a dose having at least about 10micrograms of chromium per day; c) then re-evaluating the individual'sblood serum LDL-cholesterol level.
 22. The method of claim 21 whereinthe administration of chromic tripicolinate is continued until there-evaluation indicates a decrease in blood serum LDL-cholesterol. 23.The method of claim 21 wherein said chromic tripicolinate is in apharmaceutically acceptable carrier and is orally ingested.
 24. A methodfor lowering blood serum LDL-cholesterol in an individual in need ofsuch reduction, comprising:a) evaluating the individual's blood serumLDL-cholesterol level; and b) then administering, over a plurality ofdays, a composition consisting essentially of a chromium ion containingcompound and a picolinate containing compound, in a manner such that thetwo can form chromic tripicolinate in the individual, and in an amountsufficient such that a dose of chromic tripicolinate having at leastabout 10 micrograms of chromium per day is provided to the individual;c) then re-evaluating the individual's blood serum LDL-cholesterol. 25.A method for the prevention of undesirable high levels of blood serumlipids in a patient in need thereof, comprising the administration tosaid patient of substantially pure chromic tripicolinate in a dosehaving at least about 10 micrograms of chromium per day.
 26. The methodof claim 25 wherein said chromic tripicolinate is in a pharmaceuticallyacceptable carrier and is orally ingested.
 27. The method of claim 25wherein said blood serum lipids are triglycerides.
 28. The method ofclaim 25 wherein said blood serum lipids are total serum cholesterol.29. The method of claim 25 wherein said blood serum lipids areLDL-cholesterol.
 30. The method of claim 1, wherein at least about 200μg chromium as chromic tripicolinate are administered each day for aplurality of days.
 31. The method of claim 17, wherein at least about200 μg of chromium as chromic tripicolinate are administered each dayfor at least about 40 days.